Antigenic probes locate binding sites for the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, aldolase and phosphofructokinase on the actin monomer in microfilaments.

The topology of the interfaces between actin monomers in microfilaments and three glycolytic enzymes (glyceraldehyde-3-phosphate dehydrogenase, aldolase and phosphofructokinase) was investigated using several specific antibodies directed against precisely located sequences in actin. A major contact area for glyceraldehyde-3-phosphate dehydrogenase was characterized in a region near residue 103. This interaction altered, by long-range conformational changes, the reactivity of antigenic epitopes in the C-terminal part of actin. The interface between actin and aldolase appeared to involve a sequence around residue 299 in the C-terminal region of actin. The interaction of phosphofructokinase, in contrast, modified the reactivity of all antibodies tested. Finally, the phosphagen kinases arginine kinase and creatine kinase showed no interaction with the microfilament.     

Anti-actin antibodies. An immunological approach to the myosin-actin and the tropomyosin-actin interfaces.

The topography of the rigor complex between subfragment-1 (S-1) of myosin and actin was investigated by using several specific antibodies directed to well-located sequences in actin. A major contact area for S-1 was characterized in the hydrophilic 18-28 constant sequence, and the variable 1-7 sequence was only found to be in close proximity to the interface. The C-terminal extremity of actin situated around Cys-374 appeared to be included in a region close to the S-1 heavy chain and the N-terminal part of actin. The interaction between tropomyosin and actin was also studied. Neither of the terminal parts of actin were involved in this interaction. Thus, the regions involved in the interactions of S-1 and tropomyosin with actin do not overlap.     

Paramyxovirus membrane protein enhances antibody production to new antigenic determinants in the actin molecule: a model for virus-induced autoimmunity.

Paramyxovirus membrane (M) protein specifically binds to cellular actin but not to bovine serum albumin or myoglobin, as determined by affinity chromatography and enzyme-linked immunosorbent assay. The binding site for M protein on actin is different from the binding sites for antiactin antibodies. The interaction of M protein with actin resulted in production of antibodies to several new antigenic sites on the actin molecule. Five rabbits immunized with actin alone produced antibodies against the N-terminal sequence (residues 1 to 39). Another five rabbits immunized with a mixture of M protein and actin produced antibodies against a C-terminal fragment and a central region as well as the N-terminal fragment. By immunoblotting with proteolytic fragments of actin, the new antigenic sites were located between amino acid residues 40 to 113, 114 to 226, and 227 to 375. Antisera taken from some patients with recent measles virus infections demonstrated antiactin antibodies to sites other than the N-terminal fragment of actin (amino acids 1 to 39). The interaction of paramyxovirus M protein with actin and the subsequent production of antibodies to new antigenic sites may serve as a model for one of the mechanisms of virus-induced autoimmunity.     

Differential subcellular distribution of tubulin epitopes in boar spermatozoa: recognition of class III beta-tubulin epitope in sperm tail

The exposure of tubulin epitopes was studied in ejaculated boar spermatozoa using a panel of four monoclonal antibodies specific to the N-terminal or C-terminal structural domains of tubulin and three monoclonal antibodies against class III beta-tubulin. The specificity of the antibodies was confirmed by immunoblotting. Immunocytochemical staining showed that antibodies discriminated between various parts of a spermatozoon, and that epitopes of class III beta-tubulin were present in the flagellum. A tubulin epitope from the C-terminal domain of beta-tubulin was detected in the triangular segment of the postacrosomal part of the sperm head. Its distribution changed after an A23187 ionophore-induced acrosome reaction, indicating that tubulin participates in the early stages of fertilization. Three monoclonal antibodies, TU-20, SDL.3D10, and TUJ1 directed against epitopes on the C-terminal end of neuron-specific class III beta-tubulin that is widely used as a neuronal marker, stained the flagella. The reactivity of TU-20 was further confirmed by absorbing the antibody with the immunizing peptide and by immunoelectron microscopy. Immunoblotting after two-dimensional electrophoresis revealed that the corresponding epitope was not present on all beta-tubulin isoforms. These results suggest that various tubulins are involved in the functional organization of the mammalian sperm flagellum and head.     

Structural variations in actins. A study of the immunological reactivity of the N-terminal region.

The antigenicity of the N-terminal region of skeletal-muscle actin was analysed. Two epitopes, corresponding to the 1-7 and 18-28 sequences, were determined. The antibodies specific for the first epitope discriminate skeletal-muscle actin from cardiac-muscle and smooth-muscle actins. The antibodies specific for the second epitope interact with all the actins tested, ranging from invertebrate to higher-vertebrate actins.     

Conformation of PrP(C) on the cell surface as probed by antibodies

We have investigated the conformation of Syrian hamster PrP(C) on the surface of transfected CHO cells by performing cross-competition experiments between a set of nine monoclonal antibody fragments (Fab) directed to defined epitopes throughout the protein. No competition was observed between antibodies recognizing epitopes located within the unstructured N-terminal portion of PrP(C) and those recognizing epitopes located within the ordered C-terminal half of the molecule. However, competition was observed between antibodies recognizing overlapping epitopes and between antibodies recognizing epitopes lying adjacent to one another in the PrP sequence. Titrating the reactivity of each Fab against cell-surface PrP(C) revealed a clear heterogeneity in the accessibility of different specific epitopes. Fab D18, recognizing sequence incorporating the first alpha-helix of PrP(C), bound the largest fraction of the cell-surface PrP population. In contrast, Fab E123, binding an epitope at the extreme N terminus of PrP, and Fab 13A5, binding an epitope in the central region of PrP, were able to recognize fewer than half the number of PrP(C) molecules bound by Fab D18. The pattern of antibody reactivity we observed may, in part, result from N-terminal truncation of a proportion of PrP(C) molecules found at the cell surface. However, truncation cannot account for the marked disparity between exposure of the Fab D18 and 13A5 epitopes, which lie adjacent in the PrP sequence. The relative inaccessibility of the 13A5 epitope likely reflects either PrP(C)-PrP(C) interaction, interaction between PrP(C) and other constituents on the cell membrane, or the existence of PrP(C) subspecies with distinct conformations.     

Production of antibodies to alpha(181-192) peptides of neuronal nicotinic acetylcholine receptor coupled to protein carriers in different orientations

The antibodies to nicotinic acetylcholine receptor alpha(181-192) synthetic peptides were elicited in rabbits and mice using the peptides conjugated to protein carriers in different orientations, either through C-terminal Cys (S-conjugates), or through amino groups (N-conjugates). S-conjugated peptides were less potent in eliciting peptide-specific antibodies compared to N-conjugates and this type of conjugation resulted in antibodies to the coupling reagent. However, the epitopes present in either S- or N-conjugated peptides appeared to be similar, indicating that amino acid residues, which form the epitope, were located in the middle part of the peptide and did not include both N- and C-terminal residues. Peptide conjugation to a protein carrier did not play a role in stabilizing the peptide conformation, but was necessary to concentrate the peptide epitopes on the carrier surface enabling bivalent antibody binding.     

Involvement of the C-terminal disulfide-bonded loop of murine leukemia virus SU protein in a postbinding step critical for viral entry

A role for the C-terminal domain (CTD) of murine leukemia virus (MuLV) Env protein in viral fusion was indicated by the potent inhibition of MuLV-induced fusion, but not receptor binding, by two rat monoclonal antibodies (MAbs) specific for epitopes in the CTD. Although these two MAbs, 35/56 and 83A25, have very different patterns of reactivity with viral isolates, determinants of both epitopes were mapped to the last C-terminal disulfide-bonded loop of SU (loop 10), and residues in this loop responsible for the different specificities of these MAbs were identified. Both MAbs reacted with a minor fraction of a truncated SU fragment terminating four residues after loop 10, indicating that while the deleted C-terminal residues were not part of these epitopes, they promoted their formation. Neither MAb recognized the loop 10 region expressed in isolated form, suggesting that these epitopes were not completely localized within loop 10 but required additional sequences located N terminal to the loop. Direct support for a role for loop 10 in fusion was provided by the demonstration that Env mutants containing an extra serine or threonine residue between the second and third positions of the loop were highly attenuated for infectivity and defective in fusion assays, despite wild-type levels of expression, processing, and receptor binding. Other mutations at positions 1 to 3 of loop 10 inhibited processing of the gPr80 precursor protein or led to increased shedding of SU, suggesting that loop 10 also affects Env folding and the stability of the interaction between SU and TM.     

Structural variations in actins. Biochemical and immunological tools for probing the structure of rabbit skeletal-muscle and bovine aortic actins.

Structural differences between skeletal-muscle and aortic actins were studied by using biochemical and immunological approaches. By using proteinase digestion we found that three regions of actin show structural differences: (a) in the C-terminal part, (b) the region around residue 227 and (c) the region around residue 167. By using antibodies specific to particular actin conformations we can discriminate between monomeric and filamentous forms of the two actins. Our results show that the minor sequence variations of the N- and C-terminal regions induce structural change in these regions, but also some long-range structural variations in other regions.     

Humoral immune response recognizes a complex set of epitopes on human papillomavirus type 6 l1 capsomers

Although epitope mapping has identified residues on the human papillomavirus (HPV) major capsid protein (L1) that are important for binding mouse monoclonal antibodies, epitopes recognized by human antibodies are not known. To map epitopes on HPV type 6 (HPV6) L1, surface-exposed loops were mutated to the corresponding sequence of HPV11 L1. HPV6 L1 capsomers had one to six regions mutated, including the BC, DE, EF, FG, and HI loops and the 139 C-terminal residues. After verifying proper conformation, hybrid capsomers were used in enzyme-linked immunosorbent assays with 36 HPV6-seropositive sera from women enrolled in a study of incident HPV infection. Twelve sera were HPV6 specific, while the remainder reacted with both HPV6 and HPV11 L1. By preadsorption studies, 6/11 of these sera were shown to be cross-reactive. Among the HPV6-specific sera there was no immunodominant epitope recognized by all sera. Six of the 12 sera recognized epitopes that contained residues from combinations of the BC, DE, and FG loops, one serum recognized an epitope that consisted partially of the C-terminal arm, and three sera recognized complex epitopes to which reactivity was eliminated by switching all five loops. Reactivity in two sera was not eliminated even with all six regions swapped. The patterns of epitope recognition did not change over time in women whose sera were examined 9 years after their first-seropositive visit.     

Disulfide cross-linking of caldesmon to actin.

Treatment of a solution of actin and smooth muscle caldesmon with 5,5'-dithiobis(2-nitrobenzoic acid) results in the formation of a disulfide cross-link between the C-terminal penultimate residue Cys-374 of actin and Cys-580 in caldesmon's C-terminal actin-binding region. Therefore, these 2 residues are close in the actin-caldesmon complex. Since myosin also binds to actin in the vicinity of Cys-374 and since caldesmon inhibits actomyosin ATPase activity by the reduction of myosin binding to actin, then the inhibition might be by caldesmon sterically hindering or blocking myosin's interaction with actin. [Ca2+]Calmodulin, which reverses the inhibition of the ATPase activity, decreases the yield of the cross-linked species, suggesting a weakening of the caldesmon-actin interaction in the cross-linked region. It is possible to maximally cross-link one caldesmon molecule/every three actin monomers, in the absence or presence of tropomyosin, clearly ruling out an elongated, end-to-end alignment of caldesmon on the actin filament in vitro, and raising the possibility that the N-terminal part of caldesmon projects out from the filament. Reaction of 5,5'-dithiobis(2-nitrobenzoic acid)-modified actin with caldesmon leads to the same disulfide cross-linked product between actin and caldesmon Cys-580, enabling the specific labeling of the other caldesmon cysteine, residue 153, in the N-terminal part of caldesmon with a spectroscopic probe.     

Monoclonal antibodies to either domain of ovotransferrin block binding to transferrin receptors on chick reticulocytes

Monoclonal antibodies produced to both chicken ovotransferrin and to the isolated N- and C-terminal half-molecule domains of ovotransferrin have been used to probe the interaction of ovotransferrin with its specific receptor on chick embryo red blood cells. Two antibodies to epitopes on the N-terminal domain and one antibody to an epitope on the C-terminal domain were able to block the binding of 125I-labeled diferric ovotransferrin to the receptor. When the cellular surface receptors were first saturated with ovotransferrin at 0 degrees C, none of these antibodies bound to the cell-associated ovotransferrin. This suggests that the antibodies are to epitopes which lie very near to, or in the regions of, the two domains which interact with receptor. The same three antibodies also blocked the binding to the receptor of ovotransferrin associated in situ from the isolated N- and C-terminal half-molecule domains. A fourth antibody did not block binding to receptor of 125I-labeled diferric ovotransferrin or the associated domains; furthermore, it was able to bind to ovotransferrin bound to the cell surface at 0 degrees C. This antibody thus appears to recognize an epitope remote from the receptor binding region of ovotransferrin. Additional evidence for the requirement of the presence of both domains of ovotransferrin to effect binding to the transferrin receptor on chick reticulocytes was obtained with a fifth antibody which recognized only the N-terminal half-molecule domain but not holo-ovotransferrin. Although this antibody had no effect on the binding of 125I-labeled ovotransferrin to cells, it blocked binding to receptor of the associated domains of ovotransferrin, presumably by inhibiting the association of the two domains.     

The von Willebrand factor-binding protein (vWbp) of Staphylococcus aureus is a coagulase

Staphylococcus aureus encodes a secreted von Willebrand factor-binding protein (vWbp) of 482 amino acids. The N-terminal part of this protein is homologous to staphylocoagulase and therefore we investigated whether vWbp has coagulating activity. Recombinant vWbp was shown to coagulate human and porcine plasma efficiently, but was less active against plasma from other species. The coagulation efficiency was concentration dependent, and could be inhibited by specific antibodies against vWbp. Furthermore, the species-specific coagulation by vWbp depended on the interaction with prothrombin. This interaction also resulted in specific cleavage of vWbp, releasing the C-terminal part from the coagulating domain.     

Enzyme immunoassay of immune complexes formed in vitro via interactions of serum antibodies with diphtheria toxin

The interaction of diphtheria toxin with serum antitoxin antibodies has been studied by enzyme immunoassay at variable ratios of the original amounts of the antigen and antibodies in the reaction mixture. Under the conditions of excess of the antibodies, the free toxin was not detected, and free antibodies accounted for 68 to 98% of the original amount of the antibodies. Under the conditions of excess of the toxin, free antibodies account for 2 to 7% of the original amount and free toxin, for 80–100% of its original level. Under the conditions where the toxin is taken in excess, and the amounts of the toxin and the antibodies are equivalent, formed immune complexes are regularly detected in the reaction mixtures. In these complexes, part of the epitopes of the toxin remains free from antibodies. The data obtained are interpreted from the viewpoint of epitope heterogeneity, bivalence of serum antibodies, and monovalence of the toxin epitopes. A new model of the toxin-antibody interaction is proposed.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 2, 2005, pp. 235–242.Original Russian Text Copyright © 2005 by Titova, Sviridov.     

Neutralising human recombinant antibodies to human cytomegalovirus glycoproteins gB and gH

A phage antibody display library of single chain fragment variable (scFv) was applied to develop anti-HCMV glycoprotein B (gB) and glycoprotein H (gH) neutralising libraries. To enrich for specific scFvs, the phage antibody was panned against cytomegalovirus epitopes derived from the N-terminal part of gB, the C-terminal part of gB and the N-terminal part of gH (NETIYNTTLKYGDV, VTSGSTKD and AASEALDPHAFHLLLNTYGR). A number of clones were differentiated by Bst N1 fingerprinting. After isolation of specific clones against each peptide, the neutralising effect of each clone was assessed by plaque reduction assay. This resulted in the isolation of eight neutralising scFv antibodies with 51-63% neutralising effects. Sequence analysis of three neutralising clones revealed the amino acids specificity changes in heavy and light chains of antibody molecules.     

Structure-function relationships for the IL 2-receptor system. I. Localization of a receptor binding site on IL 2

Interleukin 2 (IL 2) modulates the growth and differentiation of a variety of lymphocyte subclasses through its interaction with a specific cell surface receptor. Although both IL 2 and its receptor have been characterized extensively, the location of interaction sites on the two molecules is unknown. Synthetic peptides based on the IL 2 sequence were used to determine the epitopes seen by a number of antibodies reactive with IL 2, some of which inhibited the receptor binding of the factor and the proliferative response of target cells. The results indicated that inhibitory antibodies bound at either of two spatially distinct sites defined by amino acids 8-27 and 33-54. By inference, these segments may also encode distinct contact sites for receptor association. Alternatively, a single contact site may be located between the antibody epitopes in the three-dimensional configuration of the molecule. Although the data did not directly address the role of the C-terminal portion of IL 2, this preliminary localization of receptor contact sites within the N-terminal half of the molecule should prove useful in correlating structure and function during crystallographic analysis of the factor.     

Enzyme immunoassay of immune complexes formed in vitro via interactions of serum antibodies with diphtheria toxin

The interaction of diphtheria toxin with serum antitoxin antibodies has been studied by enzyme immunoassay at variable ratios of the original amounts of the antigen and antibodies in the reaction mixture. Under the conditions of excess of the antibodies, the free toxin is not detected, and free antibodies account for 68 to 98% of the original amount. Under the conditions of excess of the toxin, free antibodies account for 2 to 7% of the original amount and free toxin, for 80-100% of its original level. Under the conditions where the toxin is taken in excess, and the amounts of the toxin and the antibodies are equivalent, formed immune complexes are regularly detected in the reaction mixtures. In these complexes, part of the epitopes of the toxin remains free from antibodies. The data obtained are interpreted from the viewpoint of epitope heterogeneity, bivalency of serum antibodies, and monovalency of the toxin epitopes. A new model of the toxin-antibody interactions is proposed.     

Two desired epitopes of cTnI benefit for preparation of standardized monoclonal antibodies

Acute myocardial infarction (AMI) is one of the most severe cardiovascular diseases in humans, often resulting in unexpected death. Early detection is critical for patient survival. Sandwich ELISA is a common method for the detection of AMI. However, ELISA kits from different manufacturers can give different results, in part because of the lack of standardized epitopes. Therefore, the purpose of this study was to find two standardized epitopes. We predicted two antigen epitopes and respectively immunize mice to manufacture standardized monoclonal antibodies. Eight monoclonal antibodies were prepared. Monoclonal antibodies 7D2 and 2C3 were selected with high affinity, and their characteristics were explored. The results show that monoclonal antibodies 7D2 and 2C3 can both bind to various modified forms and complexes of cardiac troponin I (cTnI), were not cross‐reaction with related antigens of normal human serum and can be paired. Therefore, we deem epitopes 30 to 42 and 77 to 89 standardized epitopes.     

Phase variations of Francisella tularensis lipopolysaccharide in human infection and immunization

The comparative study of the specificity of antibodies in human sera after tularemia infection and immunization with live tularemia infection was carried out with the use of passive hemagglutination and immunoblotting techniques. The sera of tularemia patients contained two different types of immunoglobulins: strictly specific to the antigenic epitopes of F. tularensis Iipopolysaccharide (LPS) and strictly specific to F. tularensis subsp. novicida LPS. Such phenomenon may be due to phase variations of the antigenic structure of F. tularensis LPS in the body of a slightly susceptible host. The immune sera of vaccinated were found to contain antibodies, strictly specific only to F. tularensis LPS. At the same time in one vaccinee by the presence of pronounced postvaccinal reactions was found sharply defined interaction between serum imunoglobulins and F. tularensis subsp. novicida LPS. As the result, the data on the possibility of the antigenic modification of F. tularensis in tularemia infection in humans were obtained. At the same time antigenic epitopes, characteristic of faintly pathogenic and closely related F. tularensis novicida LPS, appeared in the structure of F. tularensis LPS.     

Structural and functional analysis of poly(ADP ribose) polymerase: an immunological study

Poly(ADP ribose) polymerase (EC 2.4.2.30) was studied using monoclonal antibodies for three different epitopes on the enzyme. The epitopes were mapped in relation to the functional domains of the protein and the inhibitory properties of the antibodies. The intranuclear and interspecies immunoreactivity of the enzyme was also investigated. The epitope of antibody 2 was mapped to the 17 kDa fragment generated by chymotryptic digestion of the C-terminal 54 kDa NAD-binding domain. Antibody 9 binds to the N-terminal 29 kDa fragment of the DNA binding domain and inhibits the enzyme activity by 80%. This antibody was used to purify poly(ADP ribose) polymerase by immunoaffinity chromatography. The third antibody binds to a central 36 kDa fragment that possesses part of the DNA-binding domain and the automodification domain. This antibody increases the enzymatic activity by 30%. An analysis of the species cross-reactivity of the antibodies was carried out by immunoblot analysis of nuclear proteins. Antibody 10 binding was detected in rat FR3T3 cells, Chinese hamster ovary cells (CHO) and epidermoid carcinoma lung human cells (CALU-1). The other two antibodies are specific for the human and bovine enzymes. Western blot analysis showed the association of poly(ADP ribose) polymerase with residual nuclear material obtained after nuclease treatment and high-salt extraction. Immunofluorescence studies with the three different monoclonals demonstrated that accessibility of the epitopes varies in the nucleus.